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Towards Gene Therapy For Duchenne Muscular Dystrophy Heart Disease
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Towards Gene Therapy For Duchenne Muscular Dystrophy Heart Disease

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Overview of my dissertation research

Overview of my dissertation research

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Towards Gene Therapy For Duchenne Muscular Dystrophy Heart Disease Towards Gene Therapy For Duchenne Muscular Dystrophy Heart Disease Presentation Transcript

  • Towards Gene Therapy for Duchenne Muscular Dystrophy Heart Disease Brian Bostick MD/PhD Student Duan Lab June 25, 2008 Department Seminar University of Missouri School of Medicine Department of Molecular Microbiology & Immunology
    • Background on Duchenne muscular dystrophy (DMD) heart disease
    • Background on gene therapy for DMD heart disease
    • How much gene or cell therapy do we need?
    • Which version of the dystrophin gene is the best?
    • When can we treat?
    Outline
  • What is Duchenne muscular dystrophy (DMD)?
    • Most common genetic muscle wasting disease affecting children.
    • Characterized by severe muscle weakness and wasting.
    • Disorder usually becomes apparent between 2 and 5 years of age.
    • Patients are wheelchair bound by the age of 10.
    • There is currently no cure and death occurs
    • in mid-twenties due to respiratory insufficiency
    • and/or heart failure.
  • DMD is caused by mutations in the dystrophin gene.
    • The dystrophin gene is located on the X chromosome.
    • It codes for the dystrophin protein, which localizes to the cell membrane.
    • Dystrophin is a critical muscle protein and acts like a “molecular shock absorber.”
  • Heart disease is a major source of morbidity and mortality in DMD.
    • Heart disease is the second leading cause of death in DMD.
    • Latent cardiomyopathy can often be detected at six years of age.
    • Virtually all patients have some degree of heart disease by the age of eighteen.
    • DMD heart disease usually results in a dilated cardiomyopathy (DCM).
  • DMD heart disease is characterized by specific changes in the electrocardiogram.
    • Characteristic Findings
      • Tachycardia
      • Shortened PR interval
        • conduction defect in AV node
      • Increase in the QRS duration
        • fibrosis in bundle branches
        • polyphasic R waves
      • Prolonged QT interval
        • Defect in ventricular repolarization
    Nigro 1990 Int J Cardiol
  • Hemodynamic Findings in Dilated Cardiomyopathy
    • The enlarged left ventricular chamber manifests as an increase in both the end diastolic and end systolic volumes.
    • Reduced systolic function
    Georgakopoulos 1998 AJP
  • Mdx mouse - a model for DMD
    • Dystrophin deficient mouse due to a nonsense mutation in exon 23
    • Arose from a spontaneous mutation in C57BL/10
    • Less severe pathology than DMD patients
      • Greater regenerative capacity
      • Slower progression
      • Near normal life span
      • Heart disease not well studied
  • Adeno-associated viral (AAV) gene therapy
    • Small non-pathogenic ssDNA virus.
    • Causes a minimal immune response.
    • Numerous serotypes with varying tissue tropisms
    • and transduction efficiencies.
    • A recently isolated serotype of AAV, serotype 9 (AAV-9)
    • raises the hope of systemic gene transfer.
    • Studies with AAV-9 have found it to have a broad transduction profile and to be capable of significant myocardial transduction after intravenous delivery.
    • Major drawback of AAV is its limited packaging capacity.
    • Morbidity and mortality from heart disease is increasing in DMD.
    • The potential of gene and/or cell therapy for DMD heart disease is largely unknown.
    • The heart may have different structural and functional needs for dystrophin.
    A cure for DMD requires rescuing both the skeletal muscle and the heart.
    • Background on Duchenne muscular dystrophy (DMD) heart disease
    • Background on gene therapy for DMD heart disease
    • How much gene or cell therapy do we need?
    • Which version of the dystrophin gene is the best?
    • When can we treat?
    Outline
  • Experimental Design 2. We then compared the dystrophin expression in the hearts of the female BL10, mdx, and carrier mice . 3. Next, we performed a comprehensive battery of anatomical, histopathological and functional studies of cardiovascular function. 4. Finally, we investigated potential mechanisms for protection. 1. We crossed C57BL/10 mice with mdx mice BL10 mdx Dystrophin +/+ or Dystrophin +/y Dystrophin -/y or Dystrophin -/- X Heterozygous Females (Carrier Mice) Dystrophin +/-
  • Carrier mice expressed 50% dystrophin in the heart in a mosaic pattern. Bostick et al 2008 Circ Res BL10 Female (Dystrophin +/+) Mdx Female (Dystrophin -/-) Carrier Female (Dystrophin +/-)
  • Carrier heart expression mimics gene or cell therapy. Bostick et al 2008 Circ Res Low Expressing Region of Carrier Heart High Expressing Region of Carrier Heart
  • 50% mosaic expression normalized structural and histopathological defects. Bostick et al 2008 Circ Res
  • ECG changes were completely prevented by 50% mosaic expression. Bostick et al 2008 Circ Res
  • Left ventricular catheterization revealed normal heart function with 50% mosaic expression. Bostick et al 2008 Circ Res
  • A small population of carrier mice exhibited focal inflammation which did not impact heart function. Bostick et al 2008 Circ Res
  • Selective expansion of dystrophin positive cells was not responsible for protection. Bostick et al 2008 Circ Res
  • Utrophin was up-regulated in dystrophin negative regions of carrier mouse heart. Bostick et al 2008 Circ Res
  • Conclusions
    • The mdx mouse accurately recapitulates DMD heart disease, albeit at a delayed progression.
    • The dilated cardiomyopathy of DMD is completely prevented by 50% mosaic dystrophin (full length) expression.
    • 50% mosaic dystrophin expression renders the dystrophic heart capable of tolerating stress.
    • Selective expansion of dystrophin positive cells is not responsible for protection.
    • Utrophin up-regulation may play a role in disease.
  • Future Directions
    • What is the role of utrophin upregulation.
    • What is the role of the skeletal muscle in modulating heart disease?
    • Is mosaic expression of truncated dystrophin genes sufficient to prevent heart disease?
    • Background on Duchenne muscular dystrophy (DMD) heart disease
    • Background on gene therapy for DMD heart disease
    • How much gene or cell therapy do we need?
    • Which version of the dystrophin gene is the best?
    • When can we treat?
    Outline
    • Heart failure is a significant cause of morbidity and mortality in Duchenne muscular dystrophy (DMD).
    • There is currently no effective treatment for DMD heart disease.
    • The 6 kb ∆H2-R19 mini-dystrophin gene has shown great promise for skeletal muscle gene therapy.
    Background
    • In this study, we set out to test the suitability of the ∆H2-R19 minigene for the treatment of DMD heart disease.
  • Experimental Design 1. Generate a heart-specific ∆ H2-R19 mini-dystrophin expression cassette 3. Characterize ∆H2-R19 mini-dystrophin expression 4. Perform comprehensive structure-function analysis of heart restricted ∆H2-R19 mini-dystrophin transgenic mdx mice 2. Create founder transgenic lines and breed to congenic mdx background (5 ~ 7 generations)
  • Transgenic expression of ∆H2-R19 mini-dystrophin utilizing the α -MHC promoter yielded cardiac specific expression. Bostick et al 2009 Molecular Therapy Southern Blot Western Blot Immunofluorescence
  • The ∆H2-R19 mini-dystrophin restored sarcolemmal integrity in the mdx heart. Bostick et al 2009 Molecular Therapy Evan’s Blue Dye Uptake Assay
  • Heart specific ∆H2-R19 mini-dystrophin prevented fibrosis in the mdx heart. Bostick et al 2009 Molecular Therapy Masson Trichrome
  • Uphill treadmill endurance was enhanced in mdx mice with heart specific ∆ H2-R19 mini-dystrophin expression. Bostick et al 2008 HMG under review
  • The ECG of ∆H2-R19 transgenic mdx mice was improved but not completely corrected. Bostick et al 2008 HMG under review
  • LV catheterization revealed a rescue of systolic function but not diastolic function. Bostick et al 2008 HMG under review
  • Overall left ventricular function was improved, but not completely rescued. Bostick et al 2009 Molecular Therapy
  • Heart specific ∆ H2-R19 mini-dystrophin restored dobutamine stress response and improved survival under stress. Bostick et al 2009 Molecular Therapy
  • Expressing ∆ H2-R19 mini-dystrophin on normal BL10 background profoundly displaces wild-type dystrophin.
  • Cardiovascular function is completely normalized with the heart specific ∆ H2-R19 mini-dystrophin expressed on wild-type BL10 background. N = 10 for 8-m-old BL10, N = 21 for 22-m-old BL10, N = 15 for 8-m-old transgenic BL10, and N = 7 for 22-m-old transgenic BL10 mice ECG LV Catheterization D
  • Conclusions
    • Heart specific ∆H2-R19 mini-dystrophin expression corrected cardiac histopathology and restored sarcolemmal integrity.
    • Heart function was improved but not completely normalized with heart specific ∆H2-R19 mini-dystrophin expression.
    • Heart specific ∆H2-R19 mini-dystrophin expression also restored the dobutamine stress response.
    • Skeletal muscle disease may play a role in modulate/exacerbating heart disease.
  • Future Directions
    • Further examine the role of skeletal muscle in modulating heart disease using heart-skeletal muscle double transgenic mice.
    • Investigate the potential of the micro-dystrophin gene for treatment of DMD heart disease .
    • Investigate the role of the dystrophin c-terminal domain in the heart.
    • Determine the importance of dystrophin for the conduction system of the heart.
    • Background on Duchenne muscular dystrophy (DMD) heart disease
    • Background on gene therapy for DMD heart disease
    • How much gene or cell therapy do we need?
    • Which version of the dystrophin gene is the best?
    • When can we treat?
    Outline
  • Experimental Design
    • Treat mdx mice with rAAV-9 microdystrophin at;
      • pre-clinical stage
      • symptomatic stage
    1. Generate rAAV-9 carrying the microdystrophin gene
    • Examine mice 3-6 months after treatment for signs of disease
  • AAV-9 microdystrophin efficiently transduces the heart at both the latent and symptomatic stage . Bostick et al 2008 Human Gene Therapy Latent Stage Symptomatic Stage Low Power View High Expressing Region High Expressing Region Low Expressing Region
  • Treatment with AAV-9 microdystrophin during the latent stage improves the electrocardiographic function . Bostick et al 2008 Human Gene Therapy
  • Treatment with AAV-9 microdystrophin during the symptomatic stage reduces fibrosis and improves electrocardiographic function. Symptomatic Mdx AAV Treated Mdx BL10 PR (ms) * † QT (ms) Untreated Mdx (n=5) BL10 (n=6) Treated Mdx (n=5) HR (bpm) *
  • Treatment with AAV-9 microdystrophin during the symptomatic stage improves left ventricular performance. Volume (  l) Pressure (mmHg) Untreated Mdx BL10 Treated Mdx * dP/dt Max (KmmHg/sec) * Ejection Fraction (Percent) † Stroke Volume (  l ) Untreated Mdx (n=6) BL10 (n=8) Treated Mdx (n=6) BL10 (n=8) Treated Mdx (n=6) Untreated Mdx (n=6) *
  • Conclusions
    • rAAV-9 can efficiently transduce the mouse myocardium .
    • rAAV-9 mediated microdystrophin gene therapy can treat the ECG changes during the latent phase of dystrophic cardiomyopathy.
    • rAAV-9 mediated microdystrophin gene therapy is capable of treating dystrophic cardiomyopathy during the symptomatic stages of disease.
  • Future Directions
    • Examine the potential of pre-clinical gene therapy to prevent dilated cardiomyopathy .
    • Determine the potential of rAAV-9 mediated gene therapy during the dilated/fibrotic stage of DMD heart disease.
    • Determine the potential of rAAV-9 mediated mini-dystrophin gene therapy using the dual vector technology.
  • Acknowledgements
    • Lab Members
    • Ms. Yongping Yue Mr. Nate Marschalk
    • Dr. Arka Ghosh Mr. Nick Marschalk
    • Dr. Yi Lai Ms. Amanda Shelton
    • Ms. Chun Long Mr. John Bohlmeyer
    • Dr. Dejia Li Mr. Chady Hakim
    Mentor Dr. Dongsheng Duan Collaborators Dr. Deb Fine (U. of Missouri) Dr. Jeff Chamberlain (U. of Washington) Dr. Jeff Robbins (U. of Cincinnati) Support NIH Life Sciences Training Grant Muscular Dystrophy Association NIH NIAMS Dissertation Committee Dr. Dave Pintel Dr. Mike Misfeldt Dr. Kerry McDonald Dr. Bill Fay